Processing and sustained in vitro releaseof rifampicin containing composites to enhance

the treatment of osteomyelitisNiina Ahola,1,2,* Minna Veiranto,1,3 Noora Männistö,1 Matti Karp,4 Jaana Rich,5 Alexander Efimov,4 Jukka Seppälä5

and Minna Kellomäki1,2

1Department of Biomedical Engineering; Tampere University of Technology; Tampere, Finland; 2BioMediTech; Tampere, Finland; 3Bioretec Ltd; Tampere, Finland; 4Department of

Chemistry and Bioengineering; Tampere University of Technology; Tampere, Finland; 5Department of Biotechnology and Chemical Technology; School of Chemical Technology;

Aalto University; Espoo, Finland

Keywords: drug release, antibiotic, rifampicin, biodegradable, poly(L-lactide-co-caprolactone), polylactide

The objective in this study was to develop an osteoconductive, biodegradable and rifampicin releasing bone fillingcomposite material for the treatment of osteomyelitis, a bacterial infection of bone that is very difficult and expensive totreat. The composite material will be used together with a ciprofloxacin releasing composite, because of the rapiddevelopment of resistant bacteria when rifampicin is used alone. Three composites were manufactured by twin-screwextrusion. The polymer matrix for the composites was poly(L-lactide-co-ε-caprolactone) 70/30 and all the compositescontained 8 wt% (weight percent) of rifampicin antibiotic. The b-TCP contents of the composites were 0 wt%, 50 wt%and 60 wt%. The composites were sterilized by gamma irradiation before in vitro degradation and drug release tests. Thehydrolytical degradation of the studied composites proceeded quickly and the molecular weight of the polymercomponent of the composites decreased rapidly. Rifampicin release occurred in four phases in which the high b-TCPcontent of the samples, polymer degradation and mass loss all played a role in determining the phases. The ceramiccomponent was seen to have a positive effect on the drug release. The composite with 50 wt% of b-TCP showed themost promising rifampicin release profile and it also showed activity against a common osteomyelitis causing bacteriaPseudomonas aeruginosa. A clear inhibition zone was formed in 16 h incubation. Overall, the tested materials showedgreat potential to be developed into a bone filler material for the treatment of osteomyelitis or other bone relatedinfections in combination with the ciprofloxacin releasing materials.

Introduction

This paper presents the in vitro degradation and drug releaseresults of three rifampicin releasing composite materials as well asthe effect of the most promising rifampicin releasing compositeagainst a common osteomyelitis causing bacteria Pseudomonasaeruginosa. The materials are intended for use in the treatment ofosteomyelitis or other bone related infections together with theciprofloxacin releasing composite materials presented in ouraccompanying study.1 The composites are bioabsorbable andosteoconductive due to the degradation ability of the polymermatrix (poly-L-lactide-co-ε-caprolactone) (PLCL) and the osteo-coductive properties of the ceramic filler, β-tricalcium phosphate(β-TCP). It is envisaged that these materials will enable thesimultaneous use of bone fillers that contain different antibiotics.The surgeon applying the fillers will be able to decide whichantibiotics and in which ratio to use based on the condition of thepatient and the pathogen in question. Such materials have alsobeen requested in the literature.2

Osteomyelitis, which is very challenging and expensive to treat,is a bone infection that is caused by bacteria, commonlyStaphylococcus aureus, Pseudomonas aeruginosa or Staphylococcusepidermidis.3 Traditional treatment includes surgical debridementof the infected tissue followed by long courses intravenously ororally administered antibiotics.4 Poor blood circulation in theinfected bone tissue can prevent adequate antibiotic concentra-tions in the infection site being achieved. To overcome thisproblem, local antibiotic delivery has been applied withcommercially available gentamycin releasing Septopal1 beads.The problem with this method of antibiotic delivery is that thebeads are not biodegradable and require surgical removal followedby bone grafting.5,6

Rifampicin has been used together with ciprofloxacin and otherfluoroquinolones in the treatment of osteomyelitis or other bonerelated infections. The use of rifampicin together with otherantibiotics in the treatment of Staphylococcus aureus infections hasbeen reviewed by Perlroth et al.7 In general, in in vivo and humanstudies, the combination of ciprofloxacin with rifampicin was

*Correspondence to: Niina Ahola; Email: niina.ahola@tut.fiSubmitted: 08/08/12; Revised: 10/13/12; Accepted: 11/06/12http://dx.doi.org/10.4161/biom.22793

REPORT

Biomatter 2:4, 213–225; October/November/December 2012; G 2012 Landes Bioscience

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more effective than monotherapy especially in prosthetic deviceinfections and osteomyelitis.8 Rifampicin has also been studiedtogether with fluoroquinolones in the treatment of deep sternalwound infections and studies have shown that using rifampicintogether with fluoroquinolones improves the outcome.9 The factthat rifampicin is effective against bacterial biofilms encouragesthe use of rifampicin together with other antibiotics.7,10

Rifampicin should never be used alone because resistant bacterialstrains develop quite rapidly as a result.7,10

In most of the studies reported about the combination therapyof rifampicin with other antibiotics, the delivery route of theantibiotics has been either intravenous or oral. With localantibiotic treatment, bone tissue that lacks adequate bloodcirculation can be effectively treated and the pathogenseradicated.11-19 Previous studies have also shown that with localtreatment, the drug concentrations in the blood or other tissuesare low, at least a decade lower than in the surroundingtissues,20-22 which naturally leads to decreased side effects likenausea which has been reported often as the cause fordiscontinuation of the therapy or continuing the therapy withlower dose.8,10

In this study, the potential of the materials to release rifampicinin adequate concentrations and the degradation were only testedin vitro. There is still a need for in vivo and clinical testing of thematerials. The materials do, however, show great potential for usein the treatment of osteomyelitis and other bone related infectionsand are now ready to be tested further in vivo.

Results and Discussion

The effect of processing and sterilization on the materials. Theprocessing method used for the composite materials was twin-screw extrusion and it can be assumed that the ceramic and drugparticles were evenly distributed due to the efficient mixing in theextrusion process. The composites are denoted PLCL + R [poly(L-lactide-co-ε-caprolactone) (PLCL) with 8 wt% of rifampicin infeed], PLCL + TCP50 + R [PLCL with 50 wt% of β-tricalciumphosphate (β-TCP) and 8 wt% of rifampicin in feed] and PLCL +TCP60 + R (PLCL with 50 wt% β-TCP and 8 wt% of rifampicinin feed). Processing did not cause degradation detectable withSEC measurements. However, sterilization using gamma irra-diation with a measured dose of 29–35 kGy caused significantdegradation, as was expected.23 The average molecular weight(Mw) of the raw material was measured as 246,000 g/mol and thenumber average molecular weight (Mn) 150,000 g/mol. The Mw

of the PLCL + R decreased 30% during the sterilization stage andthe Mw of the PLCL + TCP50 + R and PLCL + TCP60 + Rdecreased 40% and 30%, respectively. The Mn decreased 40% forall the composites during the sterilization stage. The polydisper-sity of PLCL + R did not change during processing but increasedfrom 1.6 to 2.0 during sterilization. For PLCL + TCP50 + R andPLCL + TCP60 + R, the PD decreased from 1.6 to 1.5 duringprocessing and increased slightly to 1.8 for PLCL + TCP50 + Rand 1.6 for PLCL + TCP60 + R.

The residual monomer content of the raw material measuredby gas chromatography was 0.08 wt% for L-lactide monomer and

below detection limit (, 0.02 wt%) for ε-caprolactone monomer.The L-lactide and ε-caprolactone monomer contents of theprocessed samples were analyzed from two points in theprocessing batch and there were two parallel samples in both.The L-lactide monomer content decreased slightly duringprocessing. It was 0.04–0.07 mol% and the caprolactonemonomer content was below 0.02 mol% for all tested samples.Because there were no significant differences in the monomercontents of the manufactured materials, it can be assumed that themonomers did not cause differences in the hydrolytic degradationbehavior of the studied composites.24

UV measurements utilizing the isosbestic point. The UV-measurements of rifampicin proved challenging due to theoxidation of rifampicin to rifampicin quinone in aqueoussolutions and in the presence of atmospheric oxygen. This canbe seen as a change in the UV-spectrum of a rifampicin solutionas well as a change in the color of the solution.25 Rifampicinquinone has a UV-spectrum partly similar to rifampicin and alsohas antibacterial properties.25 The fact that rifampicin quinonedegrades further to other compounds that do not have absorbancein the UV/V is area naturally affects the accuracy of this method.Part of the rifampicin is undetected if it has already degraded. Theaccuracy is highest when the measurements are made at shortintervals, not letting the dissolution medium stay unchanged forlong periods. However, as the release test period in this study waslong, part of the rifampicin that had oxidized to rifampicinquinone, had time to degrade to other compounds that do nothave absorptivity in the UV/V is area, even if the measurementswere performed in short intervals. It has also been reported thatrifampicin degrades more in solutions with low rifampicinconcentration.26 On the other hand, Le Guellec et al. suggestpossible in vivo stabilization of the molecule.27

In the later stages of the release test period, it was noticed thatthe degradation products of the polymer matrix induce a broadUV-peak at the beginning of the scanned area (200–220 nm).This broad peak, however, did not significantly interfere with theuse of the isosbestic point at 226 nm.

Rifampicin release from the materials. The measured initialrifampicin contents were 6.5 wt% for PLCL + R, 7.9 wt% forPLCL + TCP50 + R and 7.8 wt% for PLCL + TCP60 + R.

The cumulative release of rifampicin from the studied materialsis presented in Figure 1A. It can be seen that the release occurredin four phases and that the β-TCP content of the composites hada significant effect on the rifampicin release. Rifampicin releasefrom the PLCL + TCP60 + R was faster than from the PLCL +TCP50 + R and reached 80% in 130 d. Rifampicin release fromthe PLCL + TCP50 + R was slower but closer to zero order releasethat is the desirable release profile in this case. After 150 d, therelease slowed down. The rifampicin release from PLCL + Rshowed a lag phase at the beginning of the test series that lastedabout 50 d, during which time the release of the drug was veryslow. When compared with the ciprofloxacin release,1 the lagphase lasted longer. This is likely due to the fact that therifampicin molecule is larger than ciprofloxacin, and this causesslower diffusion through the polymer matrix and the polymerdegradation and mass loss have a stronger effect on the release.

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Because of the complex nature of the rifampicin release, thephases of the release should be considered separately. There areseveral factors affecting the drug release in this case including:the geometry of the device (cylindrical with a rough surface),high β-TCP content that introduces porosity to the matrix, theproperties of the polymer change as it degrades and mass loss.The acceleration of the drug release around the inflectionpoints in the cumulative release curves (Fig. 1A) can beeasily seen in Figure 1B, where the daily release of rifampicinis presented. The presence of the inflection points in thecumulative release profile is characteristic for a release

mechanism that is a combination of diffusion and erosionmechanisms.28

The first phase of the release was the burst at the beginning ofthe release. It was during this phase that the rifampicin moleculesnear the surface were released. The burst in the release was largerthan what was observed for ciprofloxacin releasing pellets.1

Rifampicin is more soluble in water than ciprofloxacin whichmay be the reason for the larger burst. The second phase of therelease lasted approximately until the time point of 53 d. Duringthis phase, the release was mainly governed by diffusion and therelease from PLCL + R showed a lag phase with almost negligible

Figure 1. The cumulative (A) and daily (B) release of rifampicin (R) from composites of poly(L-lactide-co-ε-caprolactone) (PLCL) and b-tricalciumphosphate (TCP) with initial TCP contents of 0 wt%, 50 wt% and 60 wt% and rifampicin content of 8 wt%. Results shown as averages with standarddeviations (n = 5).

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release. From these observations, it can be concluded that therelease of rifampicin could not occur solely by diffusion throughthe plain polymer matrix. The addition of β-TCP to the polymermatrix accelerated the release markedly. β-TCP was likely tointroduce porosity to the composite, and thus the rifampicinrelease could occur via pore diffusion in the time period up to53 d. The composites containing both rifampicin and β-TCPcan be classified as complex monolithic dispersions because ofthe high amount of filler in the composite. In these cases, therelease of the drug is often proportional to the square root oftime, but the rate of the release is higher than what is predictedby the Higuchi model.28 The second phase of the rifampicinrelease from the composites was proportional to t1/2 with R2

having values of 0.99 for PLCL + TCP50 + R, and 0.98 forPLCL + TCP60 + R. The results were also fitted to the to thepower law kinetics equation (Eqn. 1):28,29

M

Mktt n

where Mt/M∞ is the fractional drug release, k is the release rateconstant, t is time and n the release exponent. The rifampicinrelease from PLCL + TCP50 + R and PLCL + TCP60 + R had nvalues of 0.53 and 0.44 respectively. For cylindrical geometry andFickian diffusion, the diffusion release exponent n would have avalue of 0.45. For Case II diffusion (or anomalous diffusion), then has values of 0.45 , n , 1. The result suggests that therifampicin release occurs by Case II diffusion28 from the PLCL +TCP50 and by Fickian diffusion from PLCL + TCP60 + Rduring the second phase of the release.

In the third phase, the drug release was accelerated due to theincreased permeability of the polymer caused by degradation.Additionally, the polymer erosion started to play a role inincreasing the release rate. With a non-eroding system, the drugrelease would slowly decrease because it is often proportional tothe drug concentration in the device. In the case of abiodegradable system, the increased permeability and polymererosion accelerate the release.28 Here, the third phase of the releaseof PLCL + TCP50 + R obeyed again the t1/2 kinetics very well(R2 = 0.99). The release from PLCL + TCP60 + R had at thispoint reached 60% of the total cumulative release.

There was also a fourth phase to be seen in the release of PLCL+ R and PLCL + TCP50 + R but not in the PLCL + TCP60 + R.The fourth phase of the release was also not seen in theciprofloxacin release results.1 The inflection point in the releasecurves was at the time point of 116 d. It coincided well with theacceleration of mass loss from the composites. The mass loss wasalready notable at that time and the Mw of the polymer haddecreased to the level of 9,000–14,000 g/mol. From the PLCL +TCP60 + R, the available drug for release had apparently beenreleased already at this time point and thus no phase change wasseen there.

Rifampicin remaining in the samples after the in vitro testseries. At the time point of 250 d, the drug release had decreasedto an almost negligible level and 70–85% of the total rifampicinloaded in the composites had been released. The test series wascontinued for 392 d but no notable rifampicin release was

detected. The remaining rifampicin in the composites wasmeasured after the drug release test had been terminated. Themeasurement was done in a similar way to the initial rifampicincontent measurements. The brownish red color of the pellet-shaped samples indicated that some rifampicin remained in thesamples even though drug release had ceased. There has beenevidence about rifampicin being incorporated within thecrystalline parts of a polycaprolactone polymer and this mightbe the case here although the crystals may be formed of L-lactideunits.30 The remaining portion of the initial rifampicin that wasmeasured after the termination of the test series was 10.7% forPLCL + R, 5.0% for PLCL + TCP50 + R and 3.4% for PLCL +TCP60 + R. When combined with the measured releasedrifampicin, the total rifampicin amount detected did not reach100%. The fact that rifampicin oxidizes to rifampicin quinonewhich is further degraded to compounds with no UV-absorbance,explains why part of the rifampicin seems to have disappeared.25

Unfortunately, this affects the accuracy of this method inanalyzing the released rifampicin. However, the method can beused to estimate the rifampicin release and compare thecomposites with each other. In in vivo conditions, the situationmay be different. Le Guellec et al.27 found that rifampicin stabilitywas better in rifampicin containing plasma samples taken frompatients treated with rifampicin than in plasma samples whererifampicin was added in the laboratory. This improved stabilitysuggests that rifampicin may be more stable in vivo than in vitro.

Inhibition zone testing. The effect of the rifampicin releasingcomposite containing 50 wt% of β-TCP against the commonosteomyelitis causing bacteria Pseudomonas aeruginosa was testedusing bioluminescence imaging. The results are illustrated withFigure 2, where the bioluminescence imaging results (16 hincubation) of a 6-well plate cultured with Pseudomonas aeruginosaand rifampicin containing composite pellets are shown. Theantibiotic containing composite pellets are on the lower row andcontrol pellets without antibiotics are on the top row. The resultsshow that the composite material releases rifampicin in levels highenough to eradicate Pseudomonas aeruginosa. The inhibition zonecan be seen as a dark blue area surrounding the antibiotic releasingpellets in Figure 2. The blue area indicates dead bacteria whereasthe other colors indicate still living bacteria. First signs of theforming inhibition zone were observed already after two hours ofincubation. If compared with the bioluminescence results ofciprofloxacin releasing pellets1 the inhibition zone was smallerwith rifampicin. The larger molecular weight of rifampicin mayaffect the diffusion of the antibiotic in agar and make it ratherslow, which is seen as the slow growth of the inhibition zone.However, as seen in the drug release results, the initial burst waslarger with rifampicin releasing pellets than with ciprofloxacinpellets and this is probably due to the better solubility ofrifampicin.

In vitro degradation of the composites. Molecular structure.The 1H NMR spectra were measured from eight samples (rawmaterial PLCL, plain rifampicin, PLCL + R at 0, 26 and 52 weeksand PLCL + TCP50 + R at 0, 26 and 52 weeks in vitro). The 1HNMR signals of the polymer were assigned and the data wereanalyzed in the same way as in our accompanying study.1 The

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results show a similar effect of change in the comonomer ratio ofthe copolymer as the hydrolysis proceeded that was seen withciprofloxacin antibiotic containing composites.1 The L-lactide toε-caprolactone molar ratio was increased from 68/32 of the rawmaterial and the samples at 0 weeks to 77/23 of the PLCL +TCP50 + C and 82/18 of the PLCL + C at 52 weeks. A similareffect has also been reported by In Jeong et al.31

The microstructure of the copolymer was also studied similarlyto what was presented in our accompanying study.1 It was doneaccording to Herbert32 and Fernández.33 The results of the averagesequence length calculations showed the expected results based onthe findings of the ciprofloxacin containing materials.1 The PLCLcopolymer had a block structure with more random parts inbetween. The randomness factor R had a value of 0.25 for the rawmaterial and close to that for the samples prior to in vitro testing.The R factor decreased dramatically during the degradation of thepolymer to 0.18 for both PLCL + TCP50 + R and PLCL + R at52 weeks. This decrease indicates that the more random parts ofthe copolymer degrade first and the blocky structures thatcomprise long L-lactide blocks remain in the structure. At thebeginning of the test series, the sequence lengths of L-lactide were13 and increased to 23 and 31 for PLCL + TCP50 + R and PLCL+ R at 52 weeks respectively. This supports the general

interpretation that random parts of the copolymer degrade first,and thus the average sequence length is increased. The averagesequence length of ε-caprolactone was not significantly changedduring hydrolysis for any of the tested samples and had values of5.5–7 for all the analyzed samples.

Signals of rifampicin were visible in the plain rifampicin sampleand for the composite samples only in the 0 week (after processingand sterilization) samples. Due to the rather low sensitivity ofNMR, the low contents of rifampicin in the samples from the 26-and 52-week time points were not seen in the NMR spectra. The1H spectrum of plain rifampicin was compared with the spectra ofthe 0 week samples. 1H NMR analysis reported by Cellai et al.was also used when the signals were assigned to the protons of therifampicin molecule.34

Some of the rifampicin signals overlapped with the polymersignal in the composite samples and could not be analyzed. Thesewere some of the protons in the ansa chain (protons 16, 18, 20,21, 22, 2 and 31), protons 32 of the naphtalene ring, and protons59 of the methyl group attached to the piperazinyl ring. Thesignal assignment is presented as Figure 3 and the spectra can befound as supplementary data.

The amide proton and the OH-groups of the naphthalene ringwere clearly visible at d 11.98 ppm, 13.11 ppm and 13.41 ppm,

Figure 2. Bioluminescence results of the rifampicin containing (8 wt%) composites of poly(L-lactide-co-ε-caprolactone) and 50 wt% of b-tricalciumphosphate on a bacterial culture of light emitting Pseudomonas aeruginosa. Pellets containing rifampicin are on the lower row and correspondingcomposites without rifampicin are on the top row and act as controls.

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respectively. Their chemical shifts were stable and the integralswere in good stoichiometric ratio. The protons of the methylenegroup located above the plane of the naphthalene ring (protons45) showed a signal below 0 at d -0.31 ppm in plain unprocessedrifampicin and processed PLCL + R and PLCL + TCP50 + Rsamples. The fact that this signal is identical in all the samplesproves that the ansa chain has retained its original configuration.If not, the signal of this group would have appeared elsewhere inthe spectrum. The ansa bridge protons 13, 14, 15, 17, 19, 23 and25 were clearly visible, stable when compared between thesamples, and were in good stoichimetric agreement with therifampicin structure. The signals of the protons 37, 39 and 41 ofthe methyl substituents of the ansa chain and the signals of theprotons 54 and 56 of the piperazine ring were in the shoulder of alarge polymer signal, and thus their accurate integration was notpossible. Additionally, signals of the equivalent protons 53 and 57of piperazine ring appeared at d 3.02 overlapping with signals ofthe protons 19 and 47 and thus could not be accuratelyintegrated. However, their combined integral showed that therewas no change between samples in either the positions of thesignals or their integrals.

As a conclusion of the NMR analysis of rifampicin, it canbe stated that no deviations of the original rifampicin structurewere found after the analysis of the composite samples andplain rifampicin sample. Thus, rifampicin had maintained its

original structure during the processing and sterilizationstages.

Molecular weights, mass loss and water absorption. The decreaseof the molecular weights (both Mw and Mn) was rapid (Fig. 4A),and when compared with the composites with ciprofloxacinantibiotic reported in the accompanying study,1 there were nosignificant differences in the degradation behavior. At thebeginning of the hydrolysis test series, the molecular weight ofthe composites containing antibiotics degraded more rapidly thancomposites without antibiotics,40 but the differences rapidlyleveled out. Overall, the degradation obeyed first order kineticswith k values of 1.6 � 10−3 1/h for PLCL + R, 1.4 � 10−3 1/h forPLCL + TCP50 + R, and 1.4 � 10−3 1/h for PLCL + TCP60 + R.The values are very similar to those calculated for ciprofloxacincontaining composites.1 After 52 weeks of hydrolysis at 37°C andpH 7.4, the molecular weights of all the composites had decreasedby 98% to 2% of the initial value of the raw material.

The SEC distribution plots showed emerging bimodality at 20weeks for PLCL + R and clear bimodality at 39 weeks for PLCL +TCP50 + R and PLCL + TCP60 + R. The same kind ofbimodality is present in composites with ciprofloxacin antibiotic1

beginning from the 20th week of the test series. Bimodality in theSEC distribution suggest that the blocky structure of thecopolymer, shown by the 1H NMR analysis, causes the randomparts of the copolymer to degrade first and this might cause an

Figure 3. Chemical structure, atom numbering and the 1H chemical shifts of rifampicin.

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increase in a certain part of the SEC distribution curve as theblocky parts comprising mainly L-lactide monomers remain in thepolymer structure.

The mass loss of the tested materials was small and steadyduring the first ten weeks. (Fig. 4B) During the first six weeks, themass loss was caused by the rifampicin release from the polymermatrix. After this time point, the mass loss of the polymer started.At the time point of 10 weeks, the mass loss of PLCL + Raccelerated and had already reached 50% at 26 weeks. The masslosses of PLCL + TCP50 + R and PLCL + TCP60 + R weresteady and proceeded similarly for both of the composites. The

same kind of behavior was seen in the water absorption of thetested materials. The PLCL + R showed accelerated waterabsorption after 10 weeks. When compared with the results ofciprofloxacin containing composites,1 the acceleration of the waterabsorption and mass loss started earlier. Rifampicin is likely to bemore hydrophilic due to its molecular structure, and thusincreases the water absorption of the material.

β-TCP contents of the materials. The proportion of β-TCP in thecomposites was seen to increase as polymer degradationproceeded, as shown in Figure 5. The increase in the proportionof β-TCP accelerated after 10 weeks in vitro which correlates well

Figure 4. Weight average molar weight (Mw) (A), and mass loss and water absorption (B) of the studied composites as a function of time in vitro. Thecomposites comprised of poly(L-lactide-co-ε-caprolactone) (PLCL) and b-tricalcium phosphate (TCP) and rifampicin (R) with initial TCP contents of 0 wt%,50 wt% and 60 wt% and rifampicin content of 8 wt%. Error bars in part B are not visible due to the small values of standard deviations.

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with the acceleration of the mass loss of the polymer matrix.β-TCP remained in the samples due to the very slow dissolutionof β-TCP.40

Thermal properties. The changes in the glass transitiontemperatures (Tg) of the materials during the in vitro test seriesare shown in Figure 6A. The Tg values presented in Figure 6Awere taken from the second heating to ensure comparable valuesof the samples as their thermal histories were similar. Tgs of23–25°C were measured after processing and sterilization of thecomposites. This is in agreement with the literature.35 During thedegradation test series, the Tgs of the rifampicin containingcomposites decreased until week 12. The same kind of decreasewas observed with composites with ciprofloxacin but the decreasetime period was longer, up to the 20th week.1 During the first 12weeks, the Mw of the polymer decreased dramatically, which isknown to affect the Tg of a polymer.36 Additionally, the solubilityof the drug in the polymer may play a role in the glass transitionof the polymers and may plasticize the polymer causing a decrease

in Tg. An increase in the Tg after 12 weeks in vitro was due to thechange in the comonomer ratio, seen in the 1H-NMR analysis ofthe copolymer. The proportion of ε-caprolactone comonomer inthe polymer decreased and the L-lactide increased.

Melting temperatures and melting enthalpies were analyzedfrom the first heating of the samples because during the secondheating, the melting peaks no longer appeared due to fast coolingin the DSC analysis. Most of the samples showed clear bimodalityin the melting peaks the same way which was observed forciprofloxacin containing composites.1 Melting temperatures ingeneral showed a clear increase up to the 20th week in the testseries (data not shown). They increased from the initial value of115°C to 120–126°C at 20 weeks. After that the meltingtemperatures started to decrease again and ended up at 106–109°C at 52 weeks.

Melting enthalpies, which describe the crystallinity of thepolymer, were analyzed and corrected to correspond to thecombined polymer and rifampicin part of the composites.Although absolute crystallinity values could not be calculatedbecause the theoretical value of 100% crystalline poly(L-lactide-co-ε-caprolactone) 70/30 was not available, relative changes in thecrystallinity were observed. There was a steady increase in thecrystallinity of the composites throughout the test series (Fig. 6B).This indicates an increase in crystallinity as the amorphous partsof the polymer degrade first. Furthermore, the fact that shorterpolymer chains are more easily able to rearrange themselves intocrystals has an effect on the increasing crystallinity.37 The 1HNMR study showed that there were rather long L-lactide blocksin the copolymer and their proportion increased as the moreamorphous random parts of the copolymer degraded. As thepolymer chains were broken into shorter fragments, these blocksformed crystals when they were more able to rearrange themselves.Additionally, it was observed that the composite without β-TCPshowed higher melting enthalpies than the composites withβ-TCP, especially from the 20-week time point on. Thisobservation suggests that the presence of β-TCP in the compositesmay have an inhibiting effect on the crystallization.

Microstructure. Some porosity was seen in the compositescontaining β-TCP throughout the degradation test series. Thematerial without β-TCP did not show significant porosity. Thedifferences between composites containing 50 wt% or 60 wt% ofβ-TCP were not notable. SEM micrographs showed pores ofdifferent sizes in the samples throughout the hydrolysis test series(Fig. 7A–C). Pores up to the size of 100–200 mm were observedas well as smaller pores. Additionally, the surfaces of the pellet-shaped samples were porous. (Fig. 7D–F).

Rifampicin antibiotic, or ciprofloxacin antibiotic in theaccompanying article1 were not observed to affect the porosityin any way.

Conclusion

The tested composites showed rifampicin release profile with fourphases. The composite containing 50 wt% of β-TCP had themost promising steady release profile. The β-TCP was observed toaccelerate the rifampicin release. The release from the composite

Figure 5. b-tricalcium phosphate (TCP) contents of the studiedcomposites as a function of time in vitro. The composites comprised ofpoly(L-lactide-co-ε-caprolactone) (PLCL), TCP, and rifampicin (R) withinitial TCP contents of 50 wt% (TCP50), and 60 wt% (TCP60) andrifampicin content of 8 wt%. Results shown as averages with standarddeviations (n = 5). (A) PLC + 50% TCP + rifampcin and (B) PLC + 60% TCP+ rifampcin.

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without β-TCP showed a long lag phase that was probably due tothe large size of the rifampicin molecule and low diffusivity of thedrug in the polymer. As the polymer degradation proceeded andthe permeability of the polymer increased, the rifampicin releasealso accelerated. The decrease of the molecular weights was rapidas was expected and the 1H NMR results showed a change in thecomonomer ratio toward an increasing lactide comonomercontent as degradation proceeded. Additionally, the polymerwas observed to have a rather blocky structure, having long

L-lactide blocks in the structure. The composite that contained50 wt% of β-TCP showed activity in eradicating commonosteomyelitis causing bacteria Pseudomonas aeruginosa which wasseen as development of an inhibition zone in bacterial culturesutilizing bioluminescence.

Because rifampicin should not be used alone, a ciprofloxacinreleasing composite material was developed in the accompanyingstudy1 for use with the rifampicin-releasing composite materialreported here. Both of the reported composite materials show

Figure 6. Glass transition temperatures (Tg) (A) and melting enthalpies (DHf) (B) of the copolymer in the studied composites as a function of time in vitro.The composites comprised of poly(L-lactide-co-ε-caprolactone) (PLCL) and b-tricalcium phosphate (TCP) and rifampicin (R) with initial TCP contents of0 wt%, 50 wt% and 60 wt%. Results shown as averages with standard deviations (n = 2–5).

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potential to be developed into biodegradable, antibiotic releasingand osteoconductive bone-grafting materials that can be usedtogether in the treatment of osteomyelitis and other bone relatedinfections. In vitro tests are always needed to select the optimalcomposition of the materials for further studies and according tothe results reported here, the choice is the composite with 50 wt%of β-TCP. However, preclinical followed by clinical studies areneeded to establish the effect of these materials when they areimplanted in living tissue both alone and together.

Materials and Methods

The processing and characterization methods, except UV-analysis,follow the same principles as in our article concerningciprofloxacin releasing materials.1

Materials. Medical grade poly(L-lactide-co-ε-caprolactone)(PLCL) with the comonomer ratio of 70/30 and Mw of246,000 g/mol was purchased from Purac Biomaterials.β-tricalcium phosphate (β-TCP) (granule size , 38 mm) waspurchased from Plasma Biotal Ltd. Rifampicin antibiotic(molecular structure shown in Fig. 3) was purchased fromOrion Pharma. Sörensen buffer solution was prepared accordingto the standard ISO 1581438 and the chemicals used for the buffersolution (Na2HPO4 and KH2PO4) were purchased from J.T.Baker. The chemicals used for the bioluminescence bacterialcultures were: Gentamicin sulfate (Sigma-Aldrich), Isopropyl-β-D-thiogalaktopyranoside (IPTG) (Fermentas), Trypton (Lab MLimited), Yeast extract (Lab M Limited), Sodium Chloride(Merck), Agar (Merck).

Processing and sterilization. Dried polymer, β-TCP powder,and rifampicin (72 h in vacuum at room temperature) wereprocessed into rod-shaped billets with a diameter of approximately2.5 mm with a co-rotating custom-built intermeshing twin-screwextruder (L/D ratio = 22.5) in a nitrogen atmosphere. PLCLcopolymer, β-TCP and rifampicin antibiotic were delivered to the

process with separate gravimetric screw feeders. The mixing of thecomponents took place in the extruder. A haul-off unit was usedto guide the extrudate from the die and the diameter of the billetswas fine-tuned adjusting the speed of the haul-off unit. Threedifferent composites were processed. Each composite had 8 wt%rifampicin antibiotic in feed and different β-TCP contents (0, 50and 60 wt%). These composites are denoted PLCL + R, PLCL +TCP50 + R and PLCL + TCP60 + R, respectively. Pellet-shapedsamples (length approximately 2.5 mm) were cut from the billets.Before degradation tests were performed, the samples were packedand sterilized using gamma irradiation (minimum dose 25 kGy).

Drug release study. Weighed test samples (each test sampleconsisted of 15 pellets, approximately 300 mg in total) wereplaced in brown glass bottles along with 20 ml Sörensen buffersolution. Five parallel test samples were tested for each compositematerial. The bottles were placed in an incubator shaker at 37°C.At predetermined time intervals, the buffer solution waswithdrawn from each of the bottles and replaced with freshsolution. The amount of buffer solution and the periodical changeto fresh buffer solution enabled sink conditions to be validthroughout the test series. The amount of released rifampicin wasdetermined from the buffer solution using a Unicam UV 500spectrometer (ThermoSpectronic) at isosbestic point at wave-length of 226 nm. A wavelength area from 190 to 650 nm wasscanned in order to detect possible changes in the rifampicinmolecular structure which cause deviation in the UV-spectrum.Rifampicin is known to oxidize to rifampicin quinone in mildlyalkaline aqueous solutions and in the presence of atmosphericoxygen. This can be seen as a change in the UV-spectrum of arifampicin solution as well as a change in the color of the solution.Rifampicin quinone has a partly similar UV-spectrum torifampicin and it also possesses antibacterial properties.25 At awavelength of 226 nm, an isosbestic point was found that couldbe used in the rifampicin content measurements. At this point, wecan calculate the initial rifampicin content of the solution despite

Figure 7. SEM micrographs of the composites of poly(L-lactide-co-ε-caprolactone) (PLCL), 60 wt% of b-tricalcium phosphate (TCP) and rifampicin.(A–C) fractured surfaces after 0 weeks, 26 weeks and 52 weeks in vitro respectively. (D–F) Outer surfaces after 0 weeks, 26 weeks and 52 weeks in vitro,respectively.

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the fact that it has partly oxidized to rifampicin quinone, becausethe absorbance of these compounds is the same at this wavelength.The fact that rifampicin quinone degrades further to othercompounds without UV-absorbance, may affect the accuracy ofthis method. The accuracy is at its best if the measurements aremade at short intervals, not letting the dissolution medium stayunchanged for long periods.

Inhibition zone testing using bioluminescence imaging. Theeffect of the antibiotic releasing composite materials against one ofthe common osteomyelitis causing bacteria Pseudomonas aeruginosa,which was used as a non-pathogenic model organism, was testedusing bioluminescence imaging based on the ability of geneticallyengineered bacteria to emit light. The antibiotic containingcomposite material chosen for this study was the compositecontaining 50 wt% of β-TCP (PLCL + TCP50 + R) based on themost promising drug release results. Composite of PLCL and 50 wt% β-TCP without antibiotics was used as control.

An engineered bacteria strain of Pseudomonas aeruginosa PAO-LAC carrying plasmid pUCP24GW39 was used as the biosensor.The bacteria were cultured on antibiotic plates overnight at 30°Cand 300 rpm (1 mM IPTG, 10 mg/ml gentamycin), and suitablecolonies were moved into liquid culturing in LB (5 g/l yeastextract, 10 g/l tryptone and 5 g/l NaCl). If the bacteria did notproduce luminescence as wished in the morning, 1/50 dilutionwas made in a culture tube and it was incubated at 37°C and300 rpm for three hours. The level of luminescence of the cultureswas measured by using Plate ChameleonTM multilabel counter1.001 (Hidex Ltd.), and at volume of 200 ml. Counts of 1.1–2.3� 106 were found satisfying.

Layers of LB-agar (agar 15 g/l, 2 ml) were cast into 6-well plate,and the controls and antibiotic containing composite pellets wereplaced on top of them (each pellet into its own well). Bacteriawere mixed with soft LB-agar (7.5 g/l) solution and cast on top ofthe first layers. The amount of bacterial culture was dependent onthe luminescence level. It was 350–500 ml/well. After solidifica-tion, the plate was taken to the imaging station of XenogenVivoVision IVIS1 Lumina luminescence camera (CaliperLifeSciences). Pictures were taken every 20 min for 16 h withexposure time of 30 sec. The pictures were analyzed using LivingImage1 3.1 program (Caliper LifeSciences).

In vitro degradation study. Degradation tests were conductedat 37°C in vitro following the standard ISO 15814.38 First,weighed test samples (each test sample consisted of 15 pellets)were placed in brown glass bottles along with 20 ml Sörensenbuffer solution. Five parallel test samples of each compositematerial were tested at each time point. The bottles were placed inan incubator shaker at 37°C. The pH of the buffer solution wasmeasured periodically with a calibrated pH meter and the buffersolutions were changed every two weeks at the beginning of thetest series and once a week as the degradation accelerated. Testsamples were withdrawn at predetermined time points of 2, 4, 6,8, 10, 12, 16, 20, 26, 30 and 52 weeks.

Methods of analysis. Residual monomer. The determination ofresidual L-lactide and ε-caprolactone monomer contents beforeand after processing was performed by Ramboll Analytics Oy. Theε-caprolactone and L-lactide contents were measured after

chloroform extraction of the samples using gas chromatography(DC8000, CE Instruments) and after chloroform dilution usingan FI-detector. The measuring resolution was 0.02%.

Initial drug content measurements. Samples of about 150 mgwere weighed from each manufactured composition and dissolvedin 50 ml of chloroform (J.T. Baker). The amount of rifampicin inthe chloroform solution was determined using a Unicam UV 540Spectrophotometer (ThermoSpectronic) at a maximum absorp-tion wavelength of 349 nm. The rifampicin concentration in thesolution was calculated using Beer-Lambert law and standardcurves prepared with known concentrations of rifampicin.

Mass loss, water absorption and pH. After the test samples werewithdrawn from the incubator shaker, they were rinsed twice withdistilled water and the surfaces were carefully wiped with tissuepaper. The test samples were weighed immediately after wiping.The weighed test samples were dried for at least three days atambient conditions and for one week in vacuum. Finally, the testsamples were reweighed to obtain the dry masses. The dried testsamples were stored in a desiccator for further analysis.

The mass loss was calculated as the difference between theinitial mass of the test sample and the mass of the dried testsample divided by the initial mass of the test sample. The waterabsorption was calculated as the difference between the mass ofthe wet test sample and the mass of the dried test sample dividedby the mass of the dried test sample.

Molecular weights. The molecular weights (number average,Mn, and weight average, Mw, molecular weights) and poly-dispersity (PD) of the copolymer were determined at roomtemperature by size exclusion chromatography (SEC) (WatersAssociates system equipped with a Waters 717plus autosampler, aWaters 510 HPLC solvent pump, four linear PL gel columns(104, 105, 103 and 100 Å) connected in series, and a Waters 2414differential refractometer). Chloroform (Riedel-de Haën Ag,stabilized with 1% ethanol) was used as a solvent and eluent.The samples were filtered through a 0.5 mmMillex SR (Millipore)filter. The injected volume was 200 ml and the flow rate was1.0 ml/min. Monodisperse polystyrene standards were used forprimary calibration.

Thermal properties. Thermal analysis was performed usingdifferential scanning calorimeter DSC Q1000 (TA Instruments).Nitrogen was used as the sweeping gas. To ensure the samples(4–6 mg) had a similar thermal history, they were heated twiceand cooled rapidly in between. The heating rate was 20°C/min,the cooling rate was 50°C/min and the temperature range was -60°C to +200°C. Glass transition temperatures (Tg) were obtainedfrom the second heating and melting temperatures and enthalpies(Tm and DHf respectively) were obtained from the first heatingcycle. Two to five parallel samples were tested for each compositematerial and time point. The results were analyzed usingUniversal Analysis Software and averages and standard deviationswere calculated.

Ceramic content. The β-TCP content of the test samples wasmeasured using thermogravimetric analysis (TA Instruments).Approximately 20 mg of a sample was used and the samples wereheated at a rate of 20°C/min up to 700°C. The results wereanalyzed using Universal Analysis Software. Five parallel samples

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of each composition at each time point were analyzed and theresults were calculated as averages and standard deviations.

Molecular structure. The proton spectra of the samples weremeasured using Varian Mercury 300 MHz NMR Spectrometer(Varian Associates Inc.) at room temperature. Tetramethylsilane(TMS) was used as an internal standard, and chemical shifts weremeasured relative to TMS. The 1H NMR spectra were measured instandard 5 mm tubes in deuterochloroform. The data were acquireduntil the quality of the spectrum was sufficient and the number ofscans was about 512. The proton NMR spectra of the samples wereprocessed and analyzed using SpinWorks 3.1 and ACD/Spectrussoftware. Phase correction and baseline correction were applied to allspectra. Information on the molecular composition of rifampicinand the comonomer ratio of the copolymer was obtained.

Microstructure of the samples. The microstructure of thecomposites was observed using scanning electron microscopy(Philips XL-30 SEM equipped with a LaB6 filament, Philips)with an acceleration voltage of 12.0 kV. The micrographs weretaken both on the surface of the samples and on fractured samples

that were coated with gold (Edwards S150 Sputter Coater) priorto microstructure examination.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Research collaboration with Bioretec Ltd. and financial supportfrom the Finnish Funding Agency for Technology and Innovation(TEKES) and National Graduate School of MusculoskeletalDisorders and Biomaterials (N.A. and M.V.) are gratefullyappreciated. MSc Kalle Räsänen, Raija Reinikainen, KaijaHonkavaara, Eija Ahonen and MSc Vuokko Heino are warmlythanked for technical assistance. Peter Heath is thanked forchecking the language of the manuscript.

Supplemental Materials

Supplemental materials may be found here:www.landesbioscience.com/journals/biomatter/article/22793

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